ROTADIAL: The first nanobody-based immunoassay to detect Group A Rotavirus.

ROTADIAL is a rapid nanobody (Nb)-based ELISA assay able to identify Rotavirus group A (RVA) in feces from pediatric patients. The assay is based on a sandwich of two patented llama-derived Nbs directed to the inner capsid viral protein VP6 from RVA. Nbs are directed to conformational epitopes of VP6 and recognized all human RVA strains tested, representing ideal reagents for their use in immunodiagnostic tests for RVA detection. All the steps are carried out at room temperature, bringing results in less than two hours. This assay, named ROTADIAL, was validated with a reference panel of feces from pediatric patients from Argentina. The overall sensitivity and specificity of the ROTADIAL test, when compared to a commercial test, was 100 % (100/100) and 99 % (99/100) respectively. ROTADIAL presented optimal analytical performance, being capable of detecting RVA regardless of the presence of other common human enteric infectious agents and is the first RVA-diagnostic assay developed using Nbs, worldwide.

FMD empty capsids combined with the Immunostant Particle Adjuvant -ISPA or ISA206 induce protective immunity against foot and mouth disease virus.

Foot and Mouth Disease Virus (FMDV) causes economy losses and is controlled by vaccination in many countries. Vaccine formulations based on empty capsids or Virus-Like Particles (VLPs) have the advantage of avoiding the biological hazard of using infectious FMDV, albeit are poorly immunogenic. Recently, we have described that ISPA a new Immune Stimulating Complex adjuvant, is useful to improve the response against FMD of vaccines that use inactivated virus. Now, the adjuvant effects of ISPA and ISA 206 (water/oil/water) on a VLPs-based FMD vaccine were evaluated. VLPs (strain A/Argentina/2001) were obtained in mammalian cell cultures and their elicitation of an immune response against FMDV with and without ISPA or ISA 206 was evaluated in mice as a first approach. Notably, VLPs-ISPA and VLPs-ISA 206 vaccines induced protection against viral challenge in 100 % of mice, while protection induced by VLPs alone was of 40 %. Total and neutralizing FMDV antibodies were higher in the VLPs-ISPA and VLPs-ISA 206 groups compared to the VLPs group. VLPs-ISPA induced significantly higher (p < 0.001) IgG1, IgG2a, IgG2b and IgG3 titers than the VLPs vaccine. Moreover, in comparison with non-adjuvanted VLPs, VLPs-ISPA and VLPs-ISA 206 elicited an increased virus-specific T response, including higher IFNγ+/CD8 + lymphocyte production in mice. When these vaccines were tested in calves, antibody titers reached an Expected Percentage of Protection (EPP) above 90 % in the case of the VLPs-ISPA and VLPs-ISA 206 vaccines, while, in the VLPs group, EPP reached 25 %. IFNγ levels secreted by mononuclear cells of VLP-ISPA-vaccinated cattle were significantly higher than in the VLPs group. Overall, the results demonstrate that VLPs-ISPA or VLPs-ISA 206 are promising formulations for the development of a novel FMD vaccine.

Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: a candidate antigen for a BVDV ELISA.

A simple and reliable indirect enzyme-linked immunosorbent assay for detection of antibodies directed against a major bovine viral diarrhea virus (BVDV) immunogen, the E2 glycoprotein (tE2-ELISA), has been developed using the recombinant C-terminal truncated E2 glycoprotein (tE2) expressed in a Drosophila melanogaster system. This strategy demonstrated that tE2 is secreted efficiently in the supernatant, no purification steps are necessary, it is easy to produce and carries out the post translational modifications necessary to preserve its native conformation. Preliminary analysis of 183 cattle serum samples using tE2-ELISA showed a 98% specificity and a 100% sensitivity compared with the standard homologous BVDV virus neutralization test. The results also showed that the tE2 is immunoreactive because the conformation and antigenicity of the original E2 are maintained to a large extent. To our knowledge this is the first study report of the recombinant tE2 of BVDV expressed in D. melanogaster system as an antigen for ELISA.

[Expression of foot and mouth disease virus antigens in transgenic plants]. / Expresión de antígenos del virus de la fiebre aftosa en plantas transgénicas.

Owing to its geographical distribution and its highly contagious character, the foot and mouth disease (FMD) virus is responsible for one of the most dreaded of all livestock diseases. The currently-used vaccine is polyvalent and is based on an inactivated virus. Current research on FMD vaccines focuses on the creation of vaccines that are easier and cheaper to produce, and that avoid manipulation of large quantities of virus. The use of transgenic plants to express relevant antigens has been evaluated for the purpose of vaccine production. The authors' working group has taken the FMD virus as a model to evaluate the feasibility of using transgenic plants to express viral antigens and to develop experimental vaccines. The purpose of this paper is to set forth the working group's results in the expression of FMD antigens in transgenic plants.

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2.

The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the ß-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine. The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds.

An experimental subunit vaccine based on Bluetongue virus 4 VP2 protein fused to an antigen-presenting cells single chain antibody elicits cellular and humoral immune responses in cattle, guinea pigs and IFNAR(-/-) mice.

Bluetongue virus (BTV), the causative agent of bluetongue disease (BT) in domestic and wild ruminants, is worldwide distributed. A total of 27 serotypes have been described so far, and several outbreaks have been reported. Vaccination is critical for controlling the spread of BTV. In the last years, subunit vaccines, viral vector vaccines and reverse genetic-based vaccines have emerged as new alternatives to conventional ones. In this study, we developed an experimental subunit vaccine against BTV4, with the benefit of targeting the recombinant protein to antigen-presenting cells. The VP2 protein from an Argentine BTV4 isolate was expressed alone or fused to the antigen presenting cell homing (APCH) molecule, in the baculovirus insect cell expression system. The immunogenicity of both proteins was evaluated in guinea pigs and cattle. Titers of specific neutralizing antibodies in guinea pigs and cattle immunized with VP2 or APCH-VP2 were high and similar to those induced by a conventional inactivated vaccine. The immunogenicity of recombinant proteins was further studied in the IFNAR(-/-) mouse model where the fusion of VP2 to APCH enhanced the cellular immune response and the neutralizing activity induced by VP2.

High-yield expression of a viral peptide vaccine in transgenic plants.

A high-yield production of a peptide vaccine in transgenic plants is described here. A 21-mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino-terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant-derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low-cost production of peptide vaccines.

Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants.

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae.

African swine fever (ASF) has a substantial economic impact in many African developing countries and its eradication is based only on an efficient diagnosis program because of the absence of an available vaccine. Previous data suggested the convenience of using the highly antigenic virus protein p30 as ELISA antigen for serological diagnosis of this disease. A simple and efficient method is described for producing the recombinant protein p30 from ASF virus in Trichoplusia ni larvae (cabbage looper) in order to facilitate the large-scale production of this recombinant protein in the absence of fermentation procedures. A baculovirus encoding the virus protein p30 was used to infect insect larvae, showing that recombinant protein production had a sharp optimal peak with a time of occurrence dependent on the initial virus dose inoculated to the larvae. Crude lysates of infected larvae were used without further purification as coating antigen in ELISA to analyse a limited number of sera from natural or experimentally ASF virus infected pigs. Remarkably, the recombinant protein obtained from a single infected larva was sufficient for serological diagnosis of at least 3750 serum samples. Recombinant p30 obtained by this procedure was also used in a confirmatory immunoblotting, reacting with all positive sera tested previously by ELISA. In conclusion, production of the recombinant ASF virus protein p30 in larvae should be applicable to large-scale production of diagnostic reagents for this disease in developing countries, eliminating the need for specialised facilities for tissue culture.

Improvement of the immune response to foot and mouth disease virus vaccine in calves by using Avridine as adjuvant.

The epidemiological analysis of the cattle population during the eradication plan of foot and mouth disease (FMD) in Argentina clearly indicated a higher incidence of the disease in animals within their first year of age. It is important to improve the efficacy of the vaccination in those animals. In a previous report, we have shown the effect of an immunomodulator, Avridine (Avr), in the enhancement of the immune response elicited by FMD virus (FMDV) vaccines in experimental hosts [Berinstein, A., Pérez Filgueira, M., Schudel, A., Zamorano, P., Borca, M., Sadir, A.M., 1993. Avridine and LPS from Brucella ovis: effect on the memory induced by foot-and-mouth disease virus vaccination in mice. Vaccine 11, 1295-1301]. In this report, we analyze the effect of Avr in the improvement of the anti-FMDV immune response elicited in young animals immunized with a tetravalent vaccine. The anti-FMDV antibody response was evaluated using a liquid-phase blocking sandwich ELISA (LPBE) [Smitsaart, E.N., Zanelli, M., Rivera, I., Fondevila, N., Compaired, D., Maradei, E., Bianchi, T., O'Donnell, V., Schudel, A.A., 1998. Assessment using ELISA of the herd immunity levels induced in cattle by foot and mouth disease oil vaccines. Prev. Vet. Med 33, 283-296] while the cellular response was detected using an antigen specific lymphoproliferative test [Zamorano, P., Wigdorovitz, A., Chaher, M., Fernández, F., Sadir, A., Borca, M., 1994. Localization of B and T cell epitopes on a synthetic peptide containing the major immunogenic site of FMDV O1 Campos. Virology 201, 383-387]. The results show that, while no differences were detected in the cellular response, the anti-FMDV antibody reaction was significantly (<0.05) higher in animals immunized with the immunogen containing Avr. At 90 days post vaccination, 89-100% of the animals immunized with Avr presented predicted protection (PP) higher than 82% while just 50-61% of the animals immunized with vaccine without immunomodulator presented that characteristic. Also, it is shown that the increase in the anti-FMDV antibody titre in animals immunized with the vaccine containing Avr was mediated by an increase in the levels of both IgG1 and IgG2 which presented a significative correlation with LPELISA antibodies titres. It is concluded that the addition of Avr in the FMDV vaccines improve the immune status of the calves, the cattle population that suffers the highest epidemiological risk.

A comparison of methods for measuring the antibody response in mice and cattle following vaccination against foot and mouth disease.

We present a comparison of methods for evaluating the potency of foot and mouth disease vaccine in the laboratory. The anti-FMDV antibodies (Ab) in vaccinated mice were tested by liquid phase (lp) ELISA, solid phase (sp) ELISA and virus neutralization (VN), and were compared with the Ab titres detected by lpELISA, which is the official test in Argentina for testing the potency of FMD vaccines and protection against a virulent challenge in cattle. The results demonstrated that it is possible to relate the Ab levels induced in vaccinated mice with both the Ab and protective responses elicited in cattle. Furthermore, it was found that the anti-FMDV Ab titres in mice detected by lpELISA 14 days after vaccination should be an accurate parameter for predicting the results of the challenge test in cattle. Thus, this test in mice appears to be an inexpensive and rapid alternative for testing FMD vaccines in cattle.

[Mechanisms involved in the prolonged humoral immune response: behavior of aphthous fever virus]. / Mecanismos involucrados en la respuesta inmune humoral prolongada: comportamiento del virus de la fiebre aftosa.

Foot and mouth disease (FMD) is a widespread infectious disease affecting cloven-hoofed animals with severe economic consequences. Animals infected with FMD virus (FMDV) develop an immunological status of immunity characterized by high titers of virus serotype-specific neutralizing antibodies (NAb) which persist for at least 18 months. In contrast, currently inactivated virus vaccines elicit lower antibody response for shorter periods. Protection against FMDV infection has been commonly related to the level of NAb in serum. The new generation vaccines are immunologically poor, and for this reason it is important that the immunological mechanisms are activated during the infection to potentiate the action of these vaccines. The objective of this review is to present the possible mechanisms involved in the long lasting humoral immune response after FMDV infection. The necessity of the Ag for the initiation of the response is well known, although its role in maintaining and regulating the immune response is still unclear. The continuous role of the Ag in maintaining the response was demonstrated in experiments in which Abs with different specificities for long periods of time without the administration of exogen Ag were detected. The capture and retention of Ab-Ag complex by dendritic follicular cells seems to be a important factor in the increase of the Ag production and in the generation of B memory cells. The genomic persistence is strongly related to the Ag persistence. During a persistent infection, in which the genome is maintained and able to synthesize proteins, the immune system would be continuously stimulated. The continuous liberation of soluble Ag, as a productive persistent infection, induces the B cells memory for its differentiation in Abs producing cells. The antigen presenting cells (APC) are cells which present the Ag to lymphocytes in the class II MHC context. Langerhans islets, dendritic, B and phagocytic cells form the APC group. The modulation of the function of the APC cells is very important in the self regulation of the immune system. The function on the immune response depends on the capacity of generating signals for the stimulation of T cells.

Comparison of strategies for the production of FMDV empty capsids using the baculovirus vector system.

Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C). Expression levels and assembly into empty capsids were analyzed in insect cells and larvae. We observed that the use of the single-promoter vector allowed higher levels of expression both in insect cells and larvae. Recombinant capsid proteins produced by both vectors were recognized by monoclonal antibodies (mAbs) directed against conformational epitopes of FMDV A/Arg/01 and proved to self-assemble into empty capsids (75S) and pentamers (12S) when analyzed by sucrose gradient centrifugation.

First finding of genetic and antigenic diversity in 1b-BVDV isolates from Argentina.

Infection with Bovine Viral Diarrhea Viruses (BVDV) in cattle results in a wide range of clinical manifestations, ranging from mild respiratory disease to fetal death and mucosal disease, depending on the virulence of the virus and the immune and reproductive status of the host. In this study 30 Argentinean BVDV isolates were characterized by phylogenetic analysis. The isolates were genotyped based on comparison of the 5' untranslated region (5' UTR) and the E2 gene. In both phylogenetic trees, 76% of the viruses were assigned to BVDV 1b, whereas BVDV 1a, 2a and 2b were also found. Eight of the BVDV 1b isolates were further characterized by cross-neutralization tests using guinea pig antisera and sera from bovines vaccinated with two different commercial vaccines. The results demonstrated the presence of a marked antigenic diversity among Argentinean BVDV isolates and suggest the need to incorporate BVDV 1b isolates in diagnostic strategies.

Quantitation of cytokine gene expression by real time PCR in bovine milk and colostrum cells from cows immunized with a bovine rotavirus VP6 experimental vaccine.

In a previous work, VP6 recombinant protein was produced using baculovirus system and it was evaluated in a colostrum-deprived calf model. This vaccine was able to protect calves against viral challenge without inducing neutralizing antibodies (NAb), suggesting that another immunological effectors were involved in the protection observed. In this work, groups of cows (n=4) were immunized in the last third of gestation with a bovine rotavirus (BRV) experimental vaccine and with a VP6 subunit vaccine. At birth, colostrums from vaccinated and non-vaccinated cows were processed and viable colostral mononuclear cells were obtained. With the purpose of determining the cytokine patterns generated by cells from immune secretions (colostrums and milk), a relative quantification by real time PCR was standardized. Quantitative real time PCR (qPCR) was used to determine transcript levels of IL-4, IL-6, IL-10, IL-12, IFN-γ and IFN-α from these cells. Colostral and milk mononuclear cells expressed a different cytokine transcript expression pattern regarding the vaccine used. These results demonstrated that the colostral cellular population was active and could exert its action influencing the final immune response.

VP8* antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model.

Group A rotavirus is a major leading cause of diarrhea in mammalian species worldwide. In Argentina, bovine rotavirus (BRV) is the main cause of neonatal diarrhea in calves. VP4, one of the outermost capsid proteins, is involved in various virus functions. Rotavirus infectivity requires proteolytic cleavage of VP4, giving an N-terminal non-glycosilated sialic acid-recognizing domain (VP8*), and a C-terminal fragment (VP5*) that remains associated with the virion. VP8* subunit is the major determinant of the viral infectivity and one of the neutralizing antigens. In this work, the C486 BRV VP8* protein was produced in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern blot, northern blot and western blot. VP8* was highly stable in the transplastomic leaves, and formed insoluble aggregates that were partially solubilized by sonication. The recombinant protein yield was 600 µg/g of fresh tissue (FT). Both the soluble and insoluble fractions of the VP8* plant extracts were able to induce a strong immune response in female mice as measured by ELISA and virus neutralization test. Most important, suckling mice born to immunized dams were protected against oral challenge with virulent rotavirus. Results presented here contribute to demonstrate the feasibility of using antigens expressed in transplastomic plants for the development of subunit vaccines.

Development and validation of an ELISA for quantitation of bovine viral diarrhea virus antigen in the critical stages of vaccine production.

Bovine Viral Diarrhea Virus (BVDV) is the causative agent of a worldwide disease. The virus infects bovines of all ages, causing reproductive problems and contaminating biological products of high commercial value. The large-scale production of BVDV vaccines presents the challenge of processing antigenic proteins that are highly susceptible to the processing environment. Potency testing requires the immunization of cattle in order to determine the neutralizing antibodies titers induced by the vaccine. An alternative to the in vivo test is an in vitro measurement of key viral antigens. This paper describes the development and validation of a sandwich-type indirect ELISA that is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV. Validation parameters such as repeatability, intermediate precision, and reproducibility indicated that the developed ELISA constitutes an advanced tool for evaluating the BVDV antigen throughout manufacturing and vaccine release testing.

Modulation of the antigen presentation activity in foot and mouth disease virus (FMDV) vaccines by two adjuvants: avridine and a water soluble fraction of Mycobacterium sp.

We have previously demonstrated that the presence of the antigen presenting cells (APC) is critical in the induction and maintenance of the immune response in animals infected or immunized with inactivated FMDV. The use of immunological adjuvants has been repeatedly shown to be essential for the improvement of the immunogenicity in FMDV vaccines. Specifically, we have previously shown that the addition of the synthetic lipoamide Avridine (AVR) or a water soluble fraction of Mycobacterium sp. (WSF) significantly increased the immune response and protection against FMDV challenge. Here, we study the effect of these adjuvants on the induction of APC activity in mice immunized with inactivated FMDV. Both adjuvants were able to induce a long lasting antibody response which correlates with an efficient APC activity. Experiments using sequential cell transfers showed that the presence of the APC activity is not related with the efficiency of keeping free antigen in the vaccinated host. Interestingly, APC from animals immunized with AVR as adjuvant elicited virus neutralizing antibodies, while those APC obtained from donors vaccinated using WSF as adjuvant (or just an oil emulsion) induced anti-FMDV detectable only by ELISA. The analysis of the antibody response to a well studied synthetic peptide raised evidences that indicate that this difference could be explained by a differential presentation of viral B epitopes when different adjuvants were used. These results suggest that the induction of APC should be considered as one of the critical factors in the process of improving the immunogenicity of experimental FMDV vaccines.

Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus.

Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV.